The complement alternative pathway in health and disease—activation or amplification?

Abstract

In late 2020, one of us (RAH) was approached by the late Professor Sir Peter Lachmann and invited to join him in co-editing a collection of articles focused on the alternative pathway (AP), or amplification loop (AL), of C3 activation. He had recognized that much had changed since his first proposal of “C3 tickover.” New controversies had arisen, significant advances in our understanding of complement involvement in disease had been made, and, with this, there had been a considerable stimulation of interest in complement-directed pharmaceutical intervention. Re-evaluation of the pathway was required for effective exploitation of new therapeutic opportunities. Sadly, Peter passed away while the collection was still in the content planning stage,1 but, joined by CLH and JMT, and together with Immunological Reviews, we have driven the project forward. Issue 313 is the culmination of this effort. We made a conscious decision not to make this a comprehensive collection of articles covering every aspect of the AP/AL, but instead to focus on aspects that either remain controversial, or which have re-ignited controversy, or whose understanding is still in its infancy. Thus, of the components of the AP/AL, only Factor D (FD), Properdin (P), and Factor H (FH) and its related proteins Factor H-like 1 (FHL-1) and the Factor H-related (FHRs) proteins are discussed in detail. That said, Factor B (FB) and C3 are more than adequately covered in other contributions. Component-specific contributions lead to fundamental questions regarding basic mechanisms of the AP/AL of complement. The focus then moves to diseases in which there is high probability of the AP/AL being a key driver of pathogenesis, starting with discussion of how animal studies have helped our understanding of these. Finally, the collection concludes with consideration of complement-directed therapeutics, with a focus on those that directly address AP/AL dysregulation. While Peter (Figure 1) made many contributions to our understanding of complement, that for which he is best recognised is his proposal, in 1973, of the “C3 tickover” hypothesis.2 The tickover hypothesis was elegant in its simplicity—it merely stated that C3b was continually generated in blood, but without addressing the mechanism by which this occurred. In making this hypothesis, he leaned very heavily on a marriage between his understanding of complement and, in particular, the properdin pathway as it was then known, and analysis of complement involvement in rare diseases. The C3 tickover hypothesis gave a key insight into how the AP/AL functioned in vivo, providing essential surveillance and an immediate innate immune response to invading pathogens in the absence of any adaptive immunity. It was not immediately accepted, with much effort in many laboratories spent in subsequent years in attempts to provide a more conventional mechanism for AP activation and “firing” of the AL (Figure 2). However, to quote from Pangburn's contribution later in this issue: “50 years after it was proposed nothing has replaced the tickover hypothesis, attesting to Peter Lachmann's unique insight and scientific boldness”,3 and it is for this contribution in particular that many complementologists will best remember him. At its simplest, the alternative pathway for C3 activation requires just three proteins, C3, FB, and FD (Figure 3). Proteolytic activation of C3 to C3b, or generation of C3(H2O) by hydrolysis of its internal thioester bond,3 leads to assembly of C3bB or C3(H2O)B proenzyme complexes. FB bound to C3b or to C3(H2O) is the substrate for FD, and activation by FD generates the C3bBb or C3(H2O)Bb complexes, both of which can then cleave further C3 to C3b, creating a positive amplification loop for C3b generation. Unchecked, this would rapidly lead to exhaustion of C3, and hence the system is tightly regulated by the protease FI, which inactivates C3b, generating iC3b, which can no longer bind FB and participate in amplification of C3b generation. The FI-dependent inactivation of C3b requires a cofactor and, in the fluid phase, this is primarily provided by FH. In addition to providing cofactor activity, FH also accelerates the decay of C3bBb, C3(H2O)Bb, and their precursor complexes, limiting their potential to participate in amplified C3b generation. In the fluid-phase, in healthy individuals, down-regulation, not amplification, predominates. However, C3 has a further property, shared with C4 (and the α2-macroglobulin family), of an internal thioester.4 Proteolytic activation exposes the thioester, which has a transient ability to react with hydroxyl- or amino-functions on other proteins or carbohydrates, providing a long-lived “C3-tag”.5 This initial C3b-binding to an adjacent surface is non-discriminatory, and it provides a “fixed” location on which C3b generation can be amplified. The stability of surface-bound C3 convertases is also enhanced by the recruitment of properdin, until recently regarded as the only positive regulator of the AP/AL. Clearly, such non-discriminatory deposition requires that host cells are adequately protected against damaging complement attack. This is provided both by the membrane-bound regulators, CD46 (Membrane Cofactor Protein, MCP), CD55 (Decay Accelerating Factor, DAF), and CD35 (Complement Receptor 1, CR1), and by FH recruited to polyanionic surfaces. The interaction of FH with C3b and/or polyanionic surfaces is complicated by the more recently discovered Factor H-related (FHRs) proteins.6 FHRs compete with FH in binding both to C3b and to surfaces and have been termed FH-deregulators. As such they too can act as positive regulators of the AP/AL. Their discovery also means that some of the earlier work on AP/AL activation and regulation might need to be re-interpreted to take into account their possible interference in assays. Nevertheless, despite these complexities, under normal circumstances host cells exposed in blood or plasma to a fully competent AP are fully protected against the damaging effects of amplified C3 activation and C3b deposition. In contrast, pathogens lack these regulatory mechanisms, and are subjected to an amplified AP/AL-dependent response, with ensuing recruitment of C3- and C5-dependent effector functions of complement. The importance of this “first-line” defense against infection is evidenced both by the increased infection risk in complement-deficient individuals, particularly in early years when the repertoire of adaptive responses will be limited, and by the considerable spectrum of complement evasion mechanisms that pathogens have evolved.7 In their review of FD, Sekine et al8 address a fundamental challenge that has recently given rise to some controversy, the nature of circulating FD. For the AP/AL to function as an “oven-ready” surveillance mechanism as envisaged in the tickover hypothesis, sufficient FD must circulate in the active form for its activity not to be rate limiting. Recent discoveries regarding the involvement of the mannan-activated serine protease 3 (MASP-3) in the activation of FD cast doubt on this; these are addressed in detail, with reference both to the consequences of FD deficiency and to evolutionary aspects of the AP/AL. One conclusion from their manuscript is that FD circulates in man predominantly in the active form, sufficient to enable tickover surveillance, removing doubts that had been raised over this fundamental function of complement. Related to this are recent data that suggest that FD might not be the rate-limiting enzyme of the AP/AL that it has long been assumed to be.9 The Factor H related proteins (FHRs) are the most recent “additions” to the AP/AL regulator family. In their article, de Jorge et al give a comprehensive overview of this family of proteins, discussing their relationship to each other and their functional interactions, with a timely update of current understanding of their mechanism of action. In this they draw not just on biochemical and molecular biological analysis, but also on disease associations of the different members of the family.6 They also discuss the intriguing possibility that one driver of their evolution and divergence could be a need to counter a complement evasion mechanism adopted by some pathogens, that of recruiting FH to their surface to counter AP/AL attack. In some bacteria it has been demonstrated that specific FHRs can compete with FH binding to the cell membrane, diminishing the bacterium's ability to mimic host cells. This might account for the considerable differences between FHRs in mice and humans (and other species), with FHRs evolving to counter species-specific pathogens. Despite it being the first component of the AP/AL to be purified (at that time C3’ comprised not just C3, but also many of the lytic pathway components), properdin has proven one of the most difficult components to analyze. In part, this is because of its propensity to self-aggregate during isolation, aggregated properdin having properties that are not found in the native protein.10 Pedersen and co-workers present an elegant description of properdin function, largely drawn from structural insights, with novel data.11 They also bring a structural focus to the interactions of properdin with pathogens and some of their complement-evasion strategies. The role of the unusual C-mannosyl glycosylation of tryptophan residues remains enigmatic however. This glycosylation is seen too in the thrombospondin repeats of C6, C7, C8, and C9, and it has been suggested that glycosylation plays a role in the folding and stability of the thrombospondin domain.12 It would interesting to know whether these glycans interact with MBL; recruitment of MBL by surface-bound properdin, and hence MASP-3, could provide a local environment for enhanced FD activation, ensuring that it does not become rate limiting at high C3bB density. In typically erudite fashion, Liszewski and Atkinson13 set the scene for discussion of basic AP/AL mechanisms. They start with a timely reminder that a primitive surveillance system probably first arose as an intracellular system stored in intracellular vesicles, and go on to consider its evolution, not just the role in defense against pathogens, but also in maintenance of the healthy host, as a “vacuum cleaner” removing cell debris. In recent years, a number of authors have questioned current dogma over the role that water-hydrolysed C3 (now known as C3(H2O)) plays in triggering AP/AL activation.14-16 Over 40 years on from his discovery that C3(H2O) has C3b-like properties,17 Pangburn revisits the narrative.3 In particular, he addresses, through kinetic considerations, the extremely low probability that nascent C3b generated from a fluid-phase C3(H2O)Bb C3 convertase will engage with a surface, and suggests that other facilitatory mechanisms, for example, distortion of C3 when it interacts with specific surfaces such that the thioester is exposed and can react with surface groups,18 or “trace activation” of C3 through the LP or CP C3 convertases, might play a part. Interestingly, non-canonical proteolytic activation, for example by neutrophil proteases released at inflammatory sites, is not considered. Rodriguez de Cordoba19 brings clarity to a highly complex subject, that of genetics of the AP/AL components. 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